Functional distribution of epithelial sodium channels and water channels in the amphibian skin

为了定位上皮钠通道（ENaC），我们制备了一种抗非洲爪蟾ENaC α亚基胞外环肽（αxENaC）的多克隆抗体。在沙漠蟾蜍（Bufo alvarius）中，α ENaC定位于颗粒层最表层细胞的顶端细胞膜。接下来，我们使用了一种抗体，该抗体针对从日本雨蛙腹侧皮肤中克隆的水通道蛋白（水通道蛋白-2：AQP-h2）的C-末端肽。该抗体识别一个30.0 kD的蛋白质和免疫染色主要在腹部盆腔皮肤的B. alvarius。在皮肤的其他区域（如胸部和背部皮肤）中未观察到免疫反应性，这与利用骨盆皮肤的沙漠蟾蜍的吸水行为一致。免疫反应强烈的颗粒层的上层，并定位于主细胞的顶端细胞膜。腹侧皮肤暴露于抗利尿激素（AVT，10 μ <-8>M）促进了顶膜中更强烈的免疫反应。结果表明，沙漠蟾蜍能够探测到水吸收基质中的钠离子。

To localize the epithelial sodium channel (ENaC) we raised a polyclonal antibody against a peptide in the extracellular loop of the α subunit of Xenopus ENaC (αxENaC) that had been cloned from A6 kidney cell line. In desert toads, Bufo alvarius, the α ENaC was successfully localized in the apical cell membrane of the most superficial cells in the granulosum cell layer. Next, we used an antibody against, the C-terminal peptide of a water channel protein (aquaporin-2 : AQP-h2) that had been cloned from the ventral skin of the tree frog, Hyla japonica. The antibody recognized a 30.0 kD-protein and immunostained predominantly in the abdominal pelvic skin of B.alvarius. No immunoreactivity was seen in the other area of the skin such as the pectoral and dorsal skins, which is consistent to a water absorption behavior of the desert toads that utilize the pelvic skin. The immunoreactivity was intense in the upper layer of the stratum granulosum and was localized to the apical cell membrane of the principal cells. Exposure of the ventral skin to anti-diuretic hormone (AVT, 10^<-8>M) facilitated more intense immunoreactivity in the apical membrane. Results explain that the desert toad can detect sodium ions in the substrate of water absorption.